The adenomatous polyposis coli (APC) gene is a gene isolated as a causative gene of familial adenomatous polyposis (FAP) (Kinzler, K. W. and Vogelstein, B., Cell 87: 159 (1996)). However, abnormality of the APC gene is not only reported in FAP but also in 70 to 80% of the cases of sporadic colon cancer. The onset of colon cancer is considered to be triggered by stepwise mutations of multiple genes including besides APC, such genes as K-ras, p53, and DCC. Since mutations in the APC gene are found in the earliest stage among these genes, it is considered that the abnormality of the APC gene ha's to be caused first in the onset of colon cancer.
In order to clarify the mechanism underlying carcinogenesis associated with the APC gene abnormality, it is necessary to determine the functions of the gene product, APC. APC, which is a protein about 300 kDa in size, has been reported to bind with β-catenin, glycogen synthase kinase-3β (GSK-3β), as well as DLG in cells (Rubinfeld, B. et al., Science 262: 1731 (1993); Su, L. K. et al., Science 262: 1734 (1993); Rubinfeld, B. et al., Science 272: 1023 (1996); Matsumine, A. et al., Science 272: 1020 (1996)). Regarding functions of the APC, it has been reported that the intracellular level of β-catenin is rapidly reduced when wild-type APC is expressed in colon cancer cell line SW480 having mutations in the APC gene (Munemitsu, S. et al., Proc. Natl. Acad. Sci. USA 92: 3046 (1995)). The central region containing a 7-repetitive sequence structure is essential for the function of APC and coincides with a region where mutations are found in many colon cancer cases. It has also been reported that the intracellular β-catenin level is elevated in these colon cancer cells (Munemitsu, S. et al., Proc. Natl. Acad. Sci. USA 92: 3046 (1995); Rubinfeld, B. et al., Cancer Res. 57: 4624 (1997)).
β-Catenin is also known as a membrane-skeletal protein for cell adhesion molecule cadherin and also reported to participate in the signal transduction of Wnt proteins described below (Cadigan, K. M. and Nusse, R., Genes Dev. 11: 3286 (1997)). The Wnt gene family is a large gene family which has a variety of functions in the processes of early embryogenesis and morphogenesis of animals; the family consists of about 20 types of genes in mouse and the genes are conserved among a variety of animals including African clawed frog (Xenopus laevis), fruit fly (Drosophila melanogaster), and nematoda (Caenorhabditis elegans). When Wnt binds to its receptor Frizzled, the activity of glycogen synthase kinase-3β (GSK-3β) is inhibited through an intracellular signaling molecule Dishevelled (Dsh) Since the phosphorylation of β-catenin mediated by GSK-3β causes the degradation of β-catenin, the inhibition of GSK-3β activity results in accumulation of β-catenin in cells. β-Catenin binds to a transcription factor (hereinafter abbreviated as TCF) belonging to the Lef/Tcf family to form a complex and thereby activates the TCF as a transcription factor. Thus, the accumulation of β-catenin results in the formation of the β-catenin/TCF complex, which translocates to the nucleus and thereby stimulates the transcription of target genes. Among Tcfs, Tcf-4 is specifically expressed in the epithelium of colon, and thus it is believed that β-catenin mainly forms a complex with Tcf-4 in colon cancer (Korinek, V. et al., Science 275: 1784 (1997)). In addition, it has been reported that there are some colon cancer cells and melanoma cells where the APC gene is wild-type but the β-catenin gene has mutation and is not regulated by GSK-3β (Morin, P. J. et al., Science 275: 1787 (1997); Rubinfeld, B. et al., Science 275: 1790 (1997)). It has been estimated that, β-catenin constantly accumulates in these cells, which results in transcriptional activation by the β-catenin/TCF complex.
Based on the above-described findings, β-catenin may be greatly involved in the onset of colon cancer. Therefore, a substance capable of inhibiting the function of β-catenin through the binding thereto may be associated with the onset of colon cancer and the like and may be useful for the treatment, diagnosis, and such thereof. As a molecule binding to β-catenin, a protein, Axin, has been reported which negatively regulates the signal transduction system of Wnt proteins (Zeng, L. et al., Cell 90: 181 (1997)). Axin binds to GSK-3β and thereby stimulates the phosphorylation of β-catenin (Ikeda, S. et al., EMBO J. 17: 1371 (1998)). Furthermore, it has been reported that Axin also binds to APC and β-catenin to stimulate the degradation of β-catenin and thereby reducing the level of β-catenin in cells (Kishida, S. et al., J. Biol. Chem. 273: 10823 (1998); Rubinfeld, B. et al., Current Biology 8: 573 (1998); Nakamura, T. et al., Genes Cells 3: 395 (1998)). However, no protein that binds to β-catenin and has the function to localize β-catenin into the nucleus is known.
The bc19 protein (Willis T. G. et al., Blood 91: 1871 (1998)) is a product of a gene that has been cloned from the translocation site of chromosome 1 in the CEMO-1 cell line, which was established from a patient with precursor B-cell acute lymphoblastic leukemia, and the expression level of the gene is abnormally high in CEMO-1 cells. However, its association with β-catenin, such as binding to β-catenin, remains unknown.
It is desired in the art to isolate and clarify the structure of proteins and DNA encoding the protein that are useful in the treatment and diagnosis of cancer by elucidating the mechanism of the onset of cancer (including colon cancer, wherein β-catenin plays a role) through the analysis of proteins that binds to β-catenin and regulate its function and genes encoding them.